Human Sef isoforms and methods of using same for cancer diagnosis and gene therapy

• US 20060293240A1
• Filed: 07/27/2006
• Published: 12/28/2006
• Est. Priority Date: 10/11/2004
• Status: Active Grant

• Current Assignee: TECHNION RESEARCH AND DEVELOPMENT FOUNDATION, LTD.
• Original Assignee: TECHNION RESEARCH AND DEVELOPMENT FOUNDATION, LTD.
• Inventors: Ron, Dina
• Granted Patent: US 7,629,325 B2
• Time in Patent Office: Days
• US Class Current: 514/440
• CPC Class Codes: A61K 38/1709 from mammals
• CPC Class Codes: A61K 38/1825 Fibroblast growth factor [FGF]
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 14/4703 Inhibitors; Suppressors
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C07K 2319/21 containing a His-tag
• CPC Class Codes: C07K 2319/41 containing a Myc-tag

Abstract

A method and pharmaceutical compositions useful for inhibiting the growth of solid tumors are provided. Specifically, the method is effected by administering to a subject in need thereof an agent capable of upregulating the expression level and/or activity of at least a functional portion of Sef, wherein the functional portion being capable of inhibiting RTK-mediated cell proliferation. Also provided are methods and kits for diagnosing and staging of cancer by detecting the expression level of hSef in a tissue sample, wherein a decrease in hSef expression level is indicative of cancer.

Claims

1. A method of inhibiting a growth of a solid tumor in a subject, the method comprising administering to the subject an agent capable of upregulating the expression level and/or activity of at least a functional portion of Sef, said at least a functional portion of Sef being capable of inhibiting RTK-mediated cell proliferation, wherein the agent inhibits the growth of the solid tumor in the subject.

2. The method of claim 1, wherein said RTK-mediated cell proliferation is ligand independent.

3. The method of claim 1, wherein said RTK-mediated cell proliferation is ligand-induced.

4. The method of claim 1, wherein said at least a functional portion of Sef is a polypeptide as set forth by SEQ ID NO:

6.

5. The method of claim 1, wherein said at least a functional portion of Sef is a polypeptide comprising amino acids 1-10, 267-707 and/or 288-707 of SEQ ID NO:

6.

6. The method of claim 1, wherein said upregulating is effected by at least one approach selected from the group consisting of:

(a) expressing in cells of the subject an exogenous polynucleotide encoding at least a functional portion of Sef;

(b) increasing expression of endogenous Sef in cells of the subject;

(c) increasing endogenous Sef activity in cells of the subject; and

(d) introducing an exogenous peptide and/or exogenous polypeptide including at least a functional portion of Sef to the subject.

7. The method of claim 6, wherein said exogenous polynucleotide is a nucleic acid construct comprising a polynucleotide at least 90% identical to the polynucleotide sequence set forth in SEQ ID NO:

4.

8. The method of claim 6, wherein said exogenous polynucleotide is a nucleic acid construct comprising a polynucleotide selected from the group consisting of SEQ ID NOs:

4, 8, and 9.

9. The method of claim 7, wherein said nucleic acid construct further comprises a promoter capable of directing an expression of said polynucleotide in said cells of the subject.

10. The method of claim 9, wherein said promoter is selected from the group consisting of Cytomegalovirus (CMV) promoter, simian virus (SV)-40 early promoter, SV-40 late promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus (RSV) promoter, and polyhedrin promoter.

11. The method of claim 6, wherein said Sef is a polypeptide at least 90% homologous to a polypeptide set forth by SEQ ID NO:

6.

12. The method of claim 6, wherein said Sef is a polypeptide set forth by SEQ ID NO:

6.

13. The method of claim 1, wherein said solid tumor is selected from the group consisting of ovarian carcinoma, pancreatic cancer, breast cancer, endometrial carcinoma, brain tumor, adrenal carcinoma, pituitary cancer, thyroid carcinoma, tonsillar carcinoma, spleen cancer, adenoids cancer, kidney cancer, liver cancer, testis cancer, bladder cancer, colon cancer, prostate cancer, bile duct, lung cancer, and stomach cancer.

14. The method of claim 3, wherein said ligand is selected from the group consisting of FGF, PDGF, VEGF, NGF, insulin, and EGF.

15. The method of claim 1, wherein said RTK-mediated cell proliferation is effected by inhibition of activated Erk1/2 (P-Erk1/2) within said cells.

30. The pharmaceutical composition of claim 15, wherein said RTK-mediated cell proliferation is effected by inhibition of activated Erk1/2 (P-Erk1/2) within said cells.

16. A pharmaceutical composition useful for inhibiting a growth of a solid tumor in a subject comprising, as an active ingredient, an agent capable of upregulating the expression level and/or activity of at least a functional portion of Sef, said at least a functional portion of Sef being capable of inhibiting RTK-mediated cell proliferation, and a pharmaceutically acceptable carrier.

17. The pharmaceutical composition of claim 16, wherein said RTK-mediated cell proliferation is ligand independent.

18. The pharmaceutical composition of claim 16, wherein said RTK-mediated cell proliferation is ligand-induced.

19. The pharmaceutical composition of claim 16, wherein said at least a functional portion of Sef is a polypeptide as set forth by SEQ ID NO:

6.

20. The pharmaceutical composition of claim 16, wherein said at least a functional portion of Sef is a polypeptide comprising amino acids 1-10, 267-707 and/or 288-707 of SEQ ID NO:

6.

21. The pharmaceutical composition of claim 16, wherein said upregulating is effected by at least one approach selected from the group consisting of:

(a) expressing in cells of the subject an exogenous polynucleotide encoding at least a functional portion of Sef;

(b) increasing expression of endogenous Sef in cells of the subject;

(c) increasing endogenous Sef activity in cells of the subject; and

(d) introducing an exogenous peptide and/or exogenous polypeptide including at least a functional portion of Sef to the subject.

22. The pharmaceutical composition of claim 21, wherein said exogenous polynucleotide is a nucleic acid construct comprising a polynucleotide at least 90% identical to the polynucleotide sequence set forth in SEQ ID NO:

4.

23. The pharmaceutical composition of claim 21, wherein said exogenous polynucleotide is a nucleic acid construct comprising a polynucleotide selected from the group consisting of SEQ ID NOs:

4, 8, and 9.

24. The pharmaceutical composition of claim 22, wherein said nucleic acid construct further comprises a promoter capable of directing an expression of said polynucleotide in said cells of the subject.

25. The pharmaceutical composition of claim 24, wherein said promoter is selected from the group consisting of Cytomegalovirus (CMV) promoter, simian virus (SV)-40 early promoter, SV-40 late promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus (RSV) promoter, and polyhedrin promoter.

26. The pharmaceutical composition of claim 21, wherein said Sef is a polypeptide at least 90% homologous (identical+similar) to a polypeptide set forth by SEQ ID NO:

6.

27. The pharmaceutical composition of claim 21, wherein said Sef is a polypeptide set forth by SEQ ID NO:

6.

28. The pharmaceutical composition of claim 16, wherein said solid tumor is selected from the group consisting of ovarian carcinoma, pancreatic cancer, breast cancer, endometrial carcinoma, brain tumor, adrenal carcinoma, pituitary cancer, thyroid carcinoma, tonsillar carcinoma, spleen cancer, adenoids cancer, kidney cancer, liver cancer, testis cancer, bladder cancer, colon cancer, prostate cancer, bile duct, lung cancer, and stomach cancer.

29. The pharmaceutical composition of claim 18, wherein said ligand is selected from the group consisting of FGF, PDGF, VEGF, NGF, insulin and EGF.

31. A method of diagnosing cancer in a subject in need thereof, the method comprising detecting in a tissue sample of the subject an expression level of Sef, wherein a decrease in said expression level of said Sef compared to said expression level of said Sef in an unaffected tissue is indicative of the cancer, thereby diagnosing the cancer in the subject in need thereof.

32. The method of claim 31, wherein said cancer is a solid tumor.

33. The method of claim 32, wherein said solid tumor is selected from the group consisting of breast cancer, ovarian cancer, thyroid carcinoma and prostate cancer.

34. The method of claim 31, wherein said Sef is a nucleic acid sequence of Sef.

35. The method of claim 31, wherein said Sef is an amino acid sequence of Sef.

36. The method of claim 34, wherein said detecting is effected by an RNA detection method.

37. The method of claim 35, wherein said detecting is effected by a protein detection method

38. The method of claim 36, wherein said RNA detection method is selected from the group consisting of RNA in situ hybridization, RT-PCR, in situ RT-PCR and Northern blot analysis.

39. The method of claim 37, wherein said protein detection method is selected from the group consisting of ELISA, Western blot, immunofluorescence and immunohistochemical analysis.

40. The method of claim 32, wherein said solid tumor is a primary solid tumor.

41. The method of claim 32, wherein said Sef is Sef-a and whereas said solid tumor is breast cancer or ovarian cancer.

42. The method of claim 32, wherein said Sef is Sef-b and whereas said solid tumor is thyroid carcinoma.

43. The method of claim 32, further comprising determining a malignancy of said solid tumor.

44. A kit for diagnosing cancer in a subject in need thereof, the kit comprising a reagent for detecting an expression level of Sef, wherein a decrease in said expression level of said Sef compared to said expression level of said Sef in an unaffected tissue is indicative of the cancer.

45. The kit of claim 44, wherein said cancer is a solid tumor.

46. The kit of claim 45, wherein said solid tumor is selected from the group consisting of breast cancer, ovarian cancer, thyroid carcinoma and prostate cancer.

47. The kit of claim 44, wherein said Sef is a nucleic acid sequence of Sef.

48. The kit of claim 44, wherein said Sef is an amino acid sequence of Sef.

49. The kit of claim 47, wherein said reagent is utilized for an RNA detection method.

50. The kit of claim 48, wherein said reagent is utilized in a protein detection method

51. The kit of claim 49, wherein said RNA detection method is selected from the group consisting of RNA in situ hybridization, RT-PCR, in situ RT-PCR and Northern blot analysis.

52. The kit of claim 50, wherein said protein detection method is selected from the group consisting of ELISA, Western blot, immunofluorescence and immunohistochemical analysis.

53. The kit of claim 49, wherein said reagent is an isolated nucleic acid sequence complementary to said Sef nucleic acid sequence.

54. The kit of claim 50, wherein said reagent is an antibody or antibody fragment which comprises an antigen recognition region capable of specifically binding said Sef amino acid sequence.